A versatile "pan-spike-in" internal reference genome methodology for quantitative ChIP-seq normalization

项目概述：ChIP-seq已经能够绘制许多染色质因子的基因组占有率， 表观遗传组蛋白修饰，与正常生长和发育以及许多疾病的发病机制有关。 人类疾病。ChIP-seq还可以测量表观基因组标记/因子占用水平的变化 在不同染色质样品之间，例如对照与药物处理的细胞。然而，准确定量 ChIP-seq需要有效和可靠的样本归一化。含有参考外源基因组的ChIP或ChIP- Orlando等人（2014）设计的Rx通过添加交联的 果蝇S2细胞（参考外源染色质）与交联的人细胞（基于每个细胞）。抗体 对外源性和实验性测试染色质的交叉反应性对于这种掺入的成功是至关重要的 approach.外源参照种果蝇（Drosophila）、C. glabrata和S. pombe不分享许多的 表观基因组/染色质因子与实验人/小鼠或S.酿酒酵母模型。此外，缺乏 “ChIP级”抗体已导致染色质因子的表位标记。为了克服抗体的交叉反应 障碍，我们假设外源性参考基因组中的染色质结合蛋白当融合到 蛋白A和G的IgG结合域（PAG标签）将与兔或小鼠中产生的抗体交叉反应 识别表观基因组/染色质因子或表位标签，因此可以作为“一对所有或泛加标” 为了正常化。在成功使用C.用于标准化的glabrata“pan spike-in”外源参照 和测量S.酿酒厂，我们 我建议现在在人类或小鼠中创建用于ChIP-Rx的果蝇S2 R+“泛加标”参考基因组 细胞我们还建议创建S。粟酒“泛刺入”参考基因组作为非致病性替代 C. glabrata和“pan spike-in”S.酿酒酵母中ChIP-Rx的参考。pombe，all in Aim 1.我们将验证 通过测量亚基依赖性染色质，将创建的“泛刺入”基因组用于标准化的效用 使用ChIP-Rx从酵母到人的Set 1 H3 K4甲基转移酶的占有率（目的2）。在目标3中，我们建议 通过测试其他免疫球蛋白结合结构域以捕获 山羊或鸡IgG。总的来说，成功完成拟议的研究将导致创造宝贵的 试剂不仅适用于表观基因组学/染色质研究人员，而且适用于更广泛的研究社区， 为ChIP-seq提供稳健和准确的标准化方法，以量化表观基因组差异 在细胞群体、治疗和基因组状态之间。使用“平底锅加标”进行的研究 这里生成的系统将产生新的机制见解，也使全球和 发现和表征表观基因组调节因子所需的局部染色质修饰， 针对它们的药物在各种人类疾病中，从发育，代谢，神经， 疾病到癌症。

Project summary: ChIP-seq has enabled the mapping of genomic occupancy of many chromatin factors and epigenetic histone modifications, linked to normal growth and development and to the pathogenesis of many human diseases. ChIP-seq can also measure changes in the occupancy levels of epigenomic marks/factors between different chromatin samples such as control vs drug-treated cells. However, accurate quantitation in ChIP-seq requires effective and reliable sample normalization. ChIP with reference exogenous genome or ChIP- Rx devised by Orlando et al (2014) provides a rigorous normalization approach via addition of crosslinked Drosophila S2 cells (reference exogenous chromatin) with crosslinked human cells on a per-cell basis. Antibody cross-reactivity to the exogenous and experimental test chromatin is critical for the success of this spike-in approach. Exogenous reference species Drosophila, C. glabrata and S. pombe do not share many of the epigenomic/chromatin factors with the experimental human/mouse or S. cerevisiae models. Moreover, lack of “ChIP grade” antibody has led to epitope tagging of chromatin factors. To overcome the antibody cross-reactivity barriers, we hypothesized that a chromatin-binding protein in the exogenous reference genome when fused to the IgG-binding domains of Protein A and G (PAG tag) will cross-react with antibodies raised in rabbit or mouse recognizing epigenomic/chromatin factors or epitope-tags, and thus can serve as an ‘one-for-all or pan spike-in’ for normalization. Based on successfully using a C. glabrata ‘pan spike-in’ exogenous reference for normalization and measurement of chromatin occupancy differences for epigenome-modulating factors in S. cerevisiae, we propose to now create a Drosophila S2R+ ‘pan spike-in’ reference genome for ChIP-Rx in human or mouse cells. We propose to also create S. pombe ‘pan spike-in’ reference genome as a non-pathogenic alternative to C. glabrata, and a ‘pan spike-in’ S. cerevisiae reference for ChIP-Rx in S. pombe, all in Aim 1. We will validate the utility of the created ‘pan-spike-in’ genomes for normalization by measuring the subunit-dependent chromatin occupancy of Set1 H3K4 methyltransferase from yeast to humans using ChIP-Rx (Aim 2). In Aim 3, we propose to diversify the ‘pan-spike-in’ normalization system by testing other immunoglobulin-binding domains to capture goat or chicken IgG. Overall, successful completion of the proposed studies will lead to the creation of valuable reagents for not only epigenomics/chromatin researchers but also to the broader research community, by providing a robust and accurate normalization methodology for ChIP-seq to quantify epigenome differences among cell populations, treatments and genomic states. The studies resulting from using the ‘pan spike-in’ system generated here will yield novel mechanistic insights and also enable accurate quantitation of global and local chromatin modifications that is needed for the discovery and characterization of epigenome regulators and for the drugs targeting them in a variety of human diseases ranging from developmental, metabolic, neurological disorders to cancers.